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Latour and McGuigan highlight work from the Derivery laboratory that describes a new method for micropatterning proteins while maintaining their activity using fibrinogen anchors., Micropatterning is a process to precisely deposit molecules, typically proteins, onto a substrate of choice with micrometer resolution. Watson et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202009063) describe an innovative yet accessible strategy to enable the reproducible micropatterning of virtually any protein while maintaining its biological activity.

Protein micropatterning allows proteins to be precisely deposited onto a substrate of choice, and is now routinely used in cell biology and in vitro reconstitution. However, a drawback of current technology is that micropatterning efficiency can be variable between proteins, and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, Fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard to micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology.

Cytokinesis in most eukaryotic cells is orchestrated by a contractile actomyosin ring. While many of the proteins involved are known, the mechanism of constriction remains unclear. Informed by existing literature and new 3D molecular details from electron cryotomography, here we develop 3D coarse-grained models of actin filaments, unipolar and bipolar myosins, actin crosslinkers, and membranes and simulate their nteractions. Exploring a matrix of possible actomyosin configurations suggested that node-based architectures ike those presently described for ring assembly result in membrane puckers not seen in EM images of real cells. Instead, the model that best matches data from fluorescence microscopy, electron cryotomography, and biochemical experiments is one in which actin filaments transmit force to the membrane through evenly-distributed, membrane-attached, unipolar myosins, with bipolar myosins in the ring driving contraction. While at this point this model is only favored (not proven), the work highlights the power of coarse-grained biophysical simulations to compare complex mechanistic hypotheses.Significance StatementIn most eukaryotes, a ring of actin and myosin drives cell division, but how the elements of the ring are arranged and constrict remain unclear. Here we use 3D coarse-grained simulations to explore various possibilities. Our simulations suggest that if actomyosin is arranged in nodes (as suggested by a popular model of ring assembly), the membrane distorts in ways not seen experimentally. Instead, actin and myosin are more ikely uniformly distributed around the ring. In the model that best fits experimental data, ring tension is generated by interactions between bipolar myosins and actin, and transmitted to the membrane via unipolar myosins. Technologically the study highlights how coarse-grained simulations can test specific mechanistic hypotheses by comparing their predicted outcomes to experimental results.

Cell division in many eukaryotes is driven by a ring containing actin and myosin. While much is known about the main proteins involved, the precise arrangement of actin filaments within the contractile machinery, and how force is transmitted to the membrane remains unclear. Here we use cryosectioning and cryo-focused ion beam milling to gain access to cryo-preserved actomyosin rings in Schizosaccharomyces pombe for direct three-dimensional imaging by electron cryotomography. Our results show that straight, overlapping actin filaments, running nearly parallel to each other and to the membrane, form a loose bundle of approximately 150 nm in diameter that “saddles” the inward-bending membrane at the leading edge of the division septum. The filaments do not make direct contact with the membrane. Our analysis of the actin filaments reveals the variability in filament number, nearest-neighbor distances between filaments within the bundle, their distance from the membrane and angular distribution with respect to the membrane.Significance StatementMost eukaryotic cells divide using a contractile actomyosin ring, but its structure is unknown. Here we use new specimen preparation methods and electron cryotomography to image constricting rings directly in 3D, in a near-native state in the model organism Schizosaccharomyces pombe. Our images reveal the arrangement of individual actin filaments within the contracting actomyosin ring.